Dr. NADEEM AHMAD SIDDIQUE

India

Development and validation of HPTLCmethod for estimation of Umbelliferone and Quercetin in Aegle marmelos formulation.

Nadeem. A. Siddique*
1Department of Pharmaceutical Chemistry, University of Hafr Al Batin, Hafr Al Batin-31991, P.O. Box-1803, Kingdom of Saudi Arabia.

Abstract

Background

Aegle marmelos L. belongs to the family rutaceae, an important medicinal plant being used in folk therapy. The fruit of A. marmelos hold considerable amount of marmelosin, umbelliferone, quercetine. Flavonoids of Aegle marmelos fruit extract inhibit the release of autacoids and prostaglandins. Considering the therapeutic importance, in the present investigation we have screened methanolic extract of four A. marmelos formulations for the quantification and estimation of umbelliferone, quercetin, total flavonoid, phenol and developed an efficient simple and precise HPTLC method to confirm its folk medicine claim. To our knowledge, this is the first ever report to analyzed the antioxidant activity and the determination of umbelliferone and quercetin (Figure 1) in A. marmelos formulations.

Methods

Preparation of sample:
Accurately 5 gm of each formulation was weighted and sonicated for 30 min and extracted with methanol (3 × 50 ml) under reflux on water bath at 600C. It was used as working sample and applied to the plate in duplicates.
The stock solutions of umbelliferone (1 mg/ml) and quercetin (1 mg/ml) were prepared in methanol. Different concentrations (0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 ?l) in increasing order were spotted in duplicates to furnish 100, 200, 300, 400, 500, and 600 ng per spot of umbelliferone and quercetin respectively. The plate was developed in the solvent system and scanned at 333 nm for umbelliferone, and at 257 nm for quercetin.
The samples were spotted on precoated silica gel aluminium plate (20 cm × 10 cm with 0.2 mm thickness; using a CAMAG Linomat V applicator. The mobile phase consisted of toluene: ethyl acetate: formic acid (6:4:1, v/v/v) used for chromatography.

Results

The standard chromatograph shows peaks of umbelliferone at Rf 0.68 ± 0.02 and at Rf 0.67 for quercetine respectively. The peak area thus obtained with different concentration of standards were treated with linear least square regression to get the regression equation y = 2309.424+1.628*x (r2 = 0.9964), which was used for quantification of umbelliferone and for quercetine y = 401.636+21.217*x in different samples (in triplicate). The average of triplicate samples were taken and reported as concentration of umbelliferone and quercetine. Maximum amount of the umbelliferone (3.413 ± 0.3 µg/100 ml) and quercetin (3.854 ± 0.4 µg/100 ml) was analyzed in formulation F4. Likewise, highest flavonoid (8.134 ? 0.044 mg/kg) and phenolic (6.145 ± 0.05 mg/kg) content were also noticed in formulation F4..

Conclusions

In the present study, quantitative estimation of umbelliferone and quercetine in Aegle marmelos formulation were carried out successfully by HPTLC method. HPTLC method is economic, reproducible and accurate for the quality control and chemical standardization of such type of formulations.